Once synthesized on ribosomes, polypeptides must be delivered to their proper destination and be folded correctly. Many of the initial steps of these processes occur co-translationally. While comparably much is known about the translocation and folding machineries, little is understood about proteins which interact with growing polypeptide-chains near the peptidyl transferase center on the ribosome. We started to search for such proteins because by acting at the interface of ribosome and cytosol they may functionally connect the nascent-chain with proteins involved in folding and/or translocation. We have purified such a protein, NAC (Nascent- polypeptide Associated Complex). NAC is not a core ribosomal protein but contracts the nascent-chain very near to the peptidyl transferase center (17aa). In this proposal we present evidence that nascent-chains do not traverse a rigid tunnel composed of core ribosomal proteins but are covered by cytosolic proteins, including NAC, on the surface of the ribosome. When sufficiently elongated, these protecting factors have decreased affinity for the nascent-chain and are removed. Thus the nascent-chain is exposed domain by domain, rather than amino acid by amino acid to the cytosol. A central problem is to understand how, of all the proteins that mediate cotranslational events, the right factors bind to the right nascent-chain domain at the right time. Specificity of these protein interactions dictated both by the distance of the nascent-chain of the domain inquestion from the peptidyl transferase center and the amino acid sequence of the domain. The importance of ribosome/nascent-chain associated cytosolic factors is revealed in experiments where their removal results in the ability to crosslink Signal Recognition Particle (SRP) to nascent-chains whether or not they contain signal peptides in the absence of cytosol, proteins lacking signal peptides can then be mistranslocated into the ER in vitro. Readdition of cytosol or purified NAC restores the specificity of SRP and the fidelity of translocation. This demonstrates that factors such as NAC are required for signal sequence=specific sorting and translocation. We propose to study the function of NAC in vivo and in vitro. Specifically we will examine NAC's role during protein folding and sorting. Another aim is to figure out basis for NAC's binding to peptides int he context of the ribosome. Experiments outlined in this proposal are designed to allow for identification of proteins interacting with NAC and functional homologs of NAC. Our long term goal is to dissect the molecular environment of the ribosomal exit site because at this critical point the cell must ensure that only the appropriate, of many potential, fates of a nascent-chain is realized.